Ricardo N. Araujo

Validation of reference genes for expression analysis in the salivary gland and the intestine of Rhodnius prolixus (Hemiptera, Reduviidae) under different experimental conditions by quantitative real-time PCR

BackgroundRhodnius prolixus is a blood-feeding insect that can transmit Trypanosoma cruzi and Trypanosoma rangeli to vertebrate hosts. Recently, genomic resources for invertebrate vectors of human pathogens have increased significantly, and R. prolixus has been one of the main species studied among the triatomines. However, the paucity of information on many of the fundamental molecular aspects of this species limits the use of the available genomic information. The present study aimed to facilitate gene expression studies by identifying the most suitable reference genes for the normalization of mRNA expression data from qPCR.ResultsThe expression stability of five candidate reference genes (18S rRNA, GAPDH, β-actin, α-tubulin and ribosomal protein L26) was evaluated by qPCR in two tissues (salivary gland and intestine) and under different physiological conditions: before and after blood feeding and after infection with T. cruzi or T. rangeli. The results were analyzed with three software programs: geNorm, NormFinder and BestKeeper. All of the evaluated candidate genes proved to be acceptable as reference genes, but some were found to be more appropriate depending on the experimental conditions. 18S, GAPDH and α-tubulin showed acceptable stability for studies in all of the tissues and experimental conditions evaluated. β-actin, one of the most widely used reference genes, was confirmed to be one of the most suitable reference genes in studies with salivary glands, but it had the lowest expression stability in the intestine after insect blood feeding. L26 was identified as the poorest reference gene in the studies performed.ConclusionsThe expression stability of the genes varies in different tissue samples and under different experimental conditions. The results provided by three statistical packages emphasize the suitability of all five of the tested reference genes in both the crop and the salivary glands with a few exceptions. The results emphasise the importance of validating reference genes for qRT-PCR analysis in R. prolixus studies.

The role of salivary nitrophorins in the ingestion of blood by the triatomine bug Rhodnius prolixus (Reduviidae: Triatominae)

To assist haematophagy, Rhodnius prolixus produces several bioactive molecules in its saliva which it injects into the host skin. The most abundant of these molecules are the nitrophorins (NPs). In this work, we reduced the expression of NP1-4 in the saliva of R. prolixus by RNAi and evaluated the subsequent feeding performance of the bugs using the cibarial pump electromyogram either on the dorsal skin or on the tail vein of the mice. NPs salivary mRNA was reduced by >99% in comparison to controls. Saliva from knockdown nymphs also presented 82% less haemproteins while the total protein was not reduced. Knockdown nymphs feeding on the skin had lower ingestion rates mainly due to the longer cumulative probing time and lower cibarial pump frequency. Another difference was that knockdown insects bit approximately 5 times more. No differences were observed between groups fed on the tail vein. When the feeding sites were compared, nymphs fed on the tail vein had higher effective ingestion rates. These findings endorse the importance of the NPs for the ability of bugs to complete the meal in a short total contact time with a low number of bites, decreasing the perception of the insect by the host.

The Role of Salivary and Intestinal Complement System Inhibitors in the Midgut Protection of Triatomines and Mosquitoes

Saliva of haematophagous arthropods contain biomolecules involved directly or indirectly with the haematophagy process, and among them are encountered some complement system inhibitors. The most obvious function for these inhibitors would be the protection of the midgut against injury by the complement. To investigate this hypothesis, Triatoma brasiliensis nymphs were forced to ingest human serum in conditions in which the protection of midgut by the inhibitors is bypassed. In these conditions, the anterior midgut epithelium was injured by the complement, causing cell death. Once some insects such as Aedes aegypti have no salivary inhibitors, we hypothesized the existence of intestinal inhibitors. The inhibitory activity was investigated in the intestine of A. aegypti as well as in the saliva and intestine of other three triatomine species (T. brasiliensis, T. infestans and Rhodnius prolixus) using an immunological method able to determine the level of deposition of some complement factors (C1q, C3b, or C4b) on the surface of complement activator molecules linked to microplates. This methodology permitted to identify which points along the activation phase of the complement cascade were inhibited. As expected, soluble contents of A. aegyptis intestine was capable to inhibit C3b deposition by the classical and alternative pathways. Saliva or soluble intestinal contents, obtained from triatomines were unable to inhibit C1q deposition by the classical pathway. C4b deposition by the classical pathway was inhibited by the intestinal contents from the three triatomines. On the other hand, only T. brasiliensis saliva inhibited C4b deposition. Both, saliva and intestinal contents from all triatomines were able to inhibit C3b deposition in the classical and alternative pathways. None of the material extracted from the intestinal cell membranes from the triatomines inhibited C3b deposition in the classical pathway. The existence of complement inhibitors may have important biological consequences which are discussed in detail.

Filling the gaps of dinosaur eggshell phylogeny: Late Jurassic Theropod clutch with embryos from Portugal

The non-avian saurischians that have associated eggshells and embryos are represented only by the sauropodomorph Massospondylus and Coelurosauria (derived theropods), thus missing the basal theropod representatives. We report a dinosaur clutch containing several crushed eggs and embryonic material ascribed to the megalosaurid theropod Torvosaurus. It represents the first associated eggshells and embryos of megalosauroids, thus filling an important phylogenetic gap between two distantly related groups of saurischians. These fossils represent the only unequivocal basal theropod embryos found to date. The assemblage was found in early Tithonian fluvial overbank deposits of the Lourinhã Formation in West Portugal. The morphological, microstructural and chemical characterization results of the eggshell fragments indicate very mild diagenesis. Furthermore, these fossils allow unambiguous association of basal theropod osteology with a specific and unique new eggshell morphology.

The feeding process of Cimex lectularius (Linnaeus 1758) and Cimex hemipterus (Fabricius 1803) on different bloodmeal sources.

The bedbugs Cimex lectularius and Cimex hemipterus are obligate hematophages in all their nymphal instars as well as in the adult stage. The efficiency with which the insects obtain blood from their hosts is directly related to their population dynamics. In the present study we compared the feeding process and salivary content in individuals of these two species when fed on different blood sources or host sites, using a cibarial pump electromyogram. Females ingested more blood than males but needed longer contact time with the host to complete the meal. The bedbug C. lectularius was more efficient than C. hemipterus in obtaining blood from mice and pigeons. With regard to the feeding site on mice, it was easier for the insects to obtain blood from the skin of the belly than that of the back. Individuals of C. hemipterus were able to maintain the cibarial pump functioning at higher frequencies for longer periods when fed on pigeons treated with anticoagulant. Although saliva from C. lectularius contained more hemeproteins and showed more anti-clotting activity its total protein content was similar to that of C. hemipterus. Overall, C. lectularius obtains a bloodmeal more efficiently from its hosts, which may have enabled this species to reach higher levels of infestation than C. hemipterus.

Long-term effects and parental RNAi in the blood feeder Rhodnius prolixus (Hemiptera; Reduviidae)

RNA interference (RNAi) has been widely employed as a useful alternative to study gene function in insects, including triatomine bugs. However, several aspects related to the RNAi mechanism and functioning are still unclear. The aim of this study is to investigate the persistence and the occurrence of systemic and parental RNAi in the triatomine bug Rhodnius prolixus. For such, the nitrophorins 1 to 4 (NP1-4), which are salivary hemeproteins, and the rhodniin, an intestinal protein, were used as targets for RNAi. The dsRNA for both molecules were injected separately into 3rd and 5th instar nymphs of R. prolixus and the knockdown (mRNA levels and phenotype) were progressively evaluated along several stages of the insects life. We observed that the NP1-4 knockdown persisted for more than 7 months after the dsRNA injection, and at least 5 months in rhodniin knockdown, passing through various nymphal stages until the adult stage, without continuous input of dsRNA. The parental RNAi was successful from the dsRNA injection in 5th instar nymphs for both knockdown targets, when the RNAi effects (mRNA levels and phenotype) were observed at least in the 2nd instar nymphs of the F1 generation. However, the parental RNAi did not occur when the dsRNA was injected in the 3rd instars. The confirmation of the long persistence and parental transmission of RNAi in R. prolixus can improve and facilitate the utilization of this tool in insect functional genomic studies.

SALO, a novel classical pathway complement inhibitor from saliva of the sand fly Lutzomyia longipalpis

Blood-feeding insects inject potent salivary components including complement inhibitors into their host’s skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the classical pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin pathway of complement. In conclusion our data shows that SALO is a specific classical pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement classical pathway-mediated pathogenic effects in human diseases.

Influence of the intestinal anticoagulant in the feeding performance of triatomine bugs (Hemiptera; Reduviidae)

Triatomines are haematophagous insects in all post-embryonic life stages. They are vectors of Trypanosoma cruzi, the causative agent of Chagas disease. Their vectorial ability is influenced by their feeding performance, which varies greatly amongst species. Recent work showed that inhibition of the coagulation process in the anterior midgut (crop) environment considerably influences the blood meal size. In this work, we performed a comparative study of the level of anticoagulant activity in the saliva and crop contents of three triatomine species -Triatoma infestans, Triatoma brasiliensis and Rhodnius prolixus - and correlated this with their feeding performance on live hosts. Moreover, the feeding parameters on a large diameter vessel influenced by the crop anticoagulants were evaluated in detail. The anticoagulant activity was significantly higher in the crop contents than in salivary glands, varying from 1.6-fold higher for R. prolixus to 70-fold higher for T. brasiliensis. Amongst the species, T. brasiliensis had the lowest crop anticoagulant activity, the lowest concentration of thrombin inhibitor, and took the longest to feed. Triatoma brasiliensis nymphs that had their intestinal anticoagulant (brasiliensin) knocked down by RNA interference had the lowest capacity to maintain cibarial pump frequency at higher levels throughout the feeding process and consequently a lower ingestion rate (mg/min), even when fed under favourable conditions (large diameter vessel). However, the feeding difficulty for brasiliensin knockdown T. brasiliensis nymphs was reversed by treating the host mice with heparin (a potent systemic anticoagulant) before blood feeding. The results indicate that crop anticoagulant activity influences modulation of the blood-pumping frequency to the intestine and significantly affects the feeding efficiency of triatomine spp. on live hosts.

Amblyomma sculptum tick saliva: α-Gal identification, antibody response and possible association with red meat allergy in Brazil

The anaphylaxis response is frequently associated with food allergies, representing a significant public health hazard. Recently, exposure to tick bites and production of specific IgE against α-galactosyl (α-Gal)-containing epitopes has been correlated to red meat allergy. However, this association and the source of terminal, non-reducing α-Gal-containing epitopes have not previously been established in Brazil. Here, we employed the α-1,3-galactosyltransferase knockout mouse (α1,3-GalT-KO) model and bacteriophage Qβ-virus like particles (Qβ-VLPs) displaying Galα1,3Galβ1,4GlcNAc (Galα3LN) epitopes to investigate the presence of α-Gal-containing epitopes in the saliva of Amblyomma sculptum, a species of the Amblyomma cajennense complex, which represents the main tick that infests humans in Brazil. We confirmed that the α-1,3-galactosyltransferase knockout animals produce significant levels of anti-α-Gal antibodies against the Galα1,3Galβ1,4GlcNAc epitopes displayed on Qβ-virus like particles. The injection of A. sculptum saliva or exposure to feeding ticks was also found to induce both IgG and IgE anti-α-Gal antibodies in α-1,3-galactosyltransferase knockout mice, thus indicating the presence of α-Gal-containing epitopes in the tick saliva. The presence of α-Gal-containing epitopes was confirmed by ELISA and immunoblotting following removal of terminal α-Gal epitopes by α-galactosidase treatment. These results suggest for the first known time that bites from the A. sculptum tick may be associated with the unknown etiology of allergic reactions to red meat in Brazil.

Different Host Complement Systems and Their Interactions with Saliva from Lutzomyia longipalpis (Diptera, Psychodidae) and Leishmania infantum Promastigotes

Background Lutzomyia longipalpis is the vector of Leishmania infantum in the New World, and its saliva inhibits classical and alternative human complement system pathways. This inhibition is important in protecting the insect´s midgut from damage by the complement. L. longipalpis is a promiscuous blood feeder and must be protected against its host’s complement. The objective of this study was to investigate the action of salivary complement inhibitors on the sera of different host species, such as dogs, guinea pigs, rats and chickens, at a pH of 7.4 (normal blood pH) and 8.15 (the midgut pH immediately after a blood meal). We also investigated the role of the chicken complement system in Leishmania clearance in the presence and absence of vector saliva. Results The saliva was capable of inhibiting classical pathways in dogs, guinea pigs and rats at both pHs. The alternative pathway was not inhibited except in dogs at a pH of 8.15. The chicken classical pathway was inhibited only by high concentrations of saliva and it was better inhibited by the midgut contents of sand flies. Neither the saliva nor the midgut contents had any effect on the avian alternative pathway. Fowl sera killed L. infantum promastigotes, even at a low concentration (2%), and the addition of L. longipalpis saliva did not protect the parasites. The high body temperature of chickens (40°C) had no effect on Leishmania viability during our assays. Conclusion Salivary inhibitors act in a species-specific manner. It is important to determine their effects in the natural hosts of Leishmania infantum because they act on canid and rodent complements but not on chickens (which do not harbour the parasite). Moreover, we concluded that the avian complement system is the probable mechanism through which chickens eliminate Leishmania and that their high body temperature does not influence this parasite.